Composition for preventing or alleviating osteoarthritis comprising chlorogenic acid derivative as active ingredient

Abstract

The present invention relates to a composition for preventing or alleviating osteoarthritis, comprising a chlorogenic acid derivative as an active ingredient. From among chlorogenic acid derivatives, 4-O-caffeoylquinic acid (4-CQA), 5-O-caffeoylquinic acid (5-CQA), and 1,3-O-caffeoylquinic acid (1,3-DiCQA) were confirmed to exhibit preventive and therapeutic effects on osteoarthritis by suppressing expression of inflammation-related factors in osteoarthritis-induced cartilage cells, suppressing expression of extracellular matrix degradation factors, and restoring expression of homeostasis-related factors in cartilage. Accordingly, these compounds are provided as preventive or therapeutic agents for osteoarthritis.

Description

Composition for preventing or improving osteoarthritis containing a chlorogenic acid derivative as an active ingredient

[0001] The present invention relates to a composition for preventing or improving osteoarthritis, comprising a chlorogenic acid derivative as an active ingredient.

[0002] Arthritis is a general term for inflammatory diseases that occur in the joints, and is classified into osteoarthritis and rheumatoid arthritis based on their pathogenesis. Osteoarthritis is a representative geriatric disease that mainly occurs in old age. The cartilage covering the ends of the bones wears away, and the progressive damage to the articular cartilage leads to damage or deformation of the joint itself, resulting in joint pain and limited joint movement. Because osteoarthritis occurs with increasing age, it is also called degenerative arthritis or degenerative joint disease. While the cause of the disease is not yet clearly identified, it is believed to be a complex combination of factors such as genetics, age, joint trauma, and obesity. In Korea, approximately 80% of people over 55 years of age and almost everyone over 75 years of age, although the degree of osteoarthritis varies, indicating a high morbidity and prevalence rate.

[0003] Because osteoarthritis releases large amounts of inflammatory substances, such as nitric oxide (NO) and prostaglandin E2 (PGE2), associated with the inflammatory response, research into the development of osteoarthritis treatments is focusing on inhibiting iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase 2), enzymes known to produce inflammatory substances. Furthermore, studies are being conducted to determine whether substances that modulate matrix metalloproteinase (MMP) activity, which promotes the degradation of collagen, a major component of the extracellular matrix (ECM) of joints, can be used to treat osteoarthritis.

[0004] Phenybutazone, a nonsteroidal anti-inflammatory drug (NSAID) commonly used for osteoarthritis, has antipyretic, analgesic, and anti-inflammatory effects. However, it is associated with gastrointestinal, renal, cardiac, hepatic, and hematologic side effects, making long-term use impossible. Furthermore, it cannot fundamentally treat osteoarthritis, necessitating the development of new treatments. Furthermore, COX-2-selective inhibitors are known to cause cardiovascular side effects, such as stroke. Therefore, for the long-term treatment of osteoarthritis, the development of novel agents that are nontoxic, highly effective, and specifically inhibit factors associated with osteoarthritis is essential.

[0005] Meanwhile, chlorogenic acid, also called caffeoylquinic acid (CQA), is a phenolic compound produced in the phenylpropanoid biosynthetic pathway of plants. It has a structure in which caffeic acid and qunic acid are linked by an ester bond, and about 15 derivative compounds exist in nature depending on the position and number of caffeic acid bonded to qunic acid. Caffeoylquinic acid (CQA) and its derivative compounds are known to have antioxidant, antibacterial, anticancer, antiviral, anti-Alzheimer's, and neuroprotective activities.

[0006] [Prior Art Literature]

[0007] [Patent Document]

[0008] Korean Patent Publication No. 10-2556530 (Published on July 14, 2023)

[0009] The purpose of the present invention is to provide compounds that exhibit preventive and therapeutic effects on osteoarthritis by inhibiting the expression of inflammatory factors in osteoarthritis-induced cartilage cells, inhibiting the expression of extracellular matrix decomposition factors, and restoring the expression of cartilage homeostasis-related factors among chlorogenic acid derivatives, as a preventive or therapeutic agent for osteoarthritis.

[0010] The present invention provides a pharmaceutical composition for preventing or treating osteoarthritis, comprising a chlorogenic acid derivative, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof as an active ingredient.

[0011] In addition, the present invention provides a health functional food composition for preventing or improving osteoarthritis, comprising a chlorogenic acid derivative, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof as an active ingredient.

[0012] According to the present invention, among chlorogenic acid derivatives, 4-O-caffeoylquinic acid (4-CQA), 5-O-caffeoylquinic acid (5-CQA), and 1,3-O-caffeoylquinic acid (1,3-DiCQA) inhibit the expression of inflammatory factors in osteoarthritis-induced chondrocytes, inhibit the expression of extracellular matrix degrading factors, and restore the expression of cartilage homeostasis-related factors, thereby demonstrating preventive and therapeutic effects on osteoarthritis. Therefore, these compounds can be provided as agents for the prevention or treatment of osteoarthritis.

[0013] Figure 1 shows the results of evaluating the cytotoxicity of 4-O-caffeoylquinic acid (4-CQA), a chlorogenic acid derivative, in chondrocytes.

[0014] Figure 2a shows the results of analyzing the effect of 4-O-caffeoylquinic acid (4-CQA), a chlorogenic acid derivative, on the expression of PTGS2 (prostaglandin-endoperoxide synthase 2) and NOS2 (nitric oxide synthase 2) genes, which are inflammation-related factors, in chondrocytes.

[0015] Figure 2b shows the results of analyzing the effect of 4-O-caffeoylquinic acid (4-CQA), a chlorogenic acid derivative, on changes in the expression of cyclooxygenase 2 (COX2) and nitric oxide synthase 2 (NOS2), which are inflammation-related factors, in chondrocytes.

[0016] Figure 3a shows the results of analyzing the effect of 4-O-caffeoylquinic acid (4-CQA), a chlorogenic acid derivative, on changes in the expression of matrix metalloproteinase-3 (MMP3) and matrix metalloproteinase-13 (MMP13) genes in chondrocytes.

[0017] Figure 3b shows the results of analyzing the effect of 4-O-caffeoylquinic acid (4-CQA), a chlorogenic acid derivative, on changes in the expression of matrix metalloproteinase-3 (MMP3) and matrix metalloproteinase-13 (MMP13) proteins in chondrocytes.

[0018] Figure 3c shows the results of analyzing the effect of 4-O-caffeoylquinic acid (4-CQA), a chlorogenic acid derivative, on the levels of matrix metalloproteinase-3 (MMP3) and matrix metalloproteinase-13 (MMP13) proteins secreted outside the cells in chondrocytes.

[0019] Figure 4a shows the results of analyzing the effect of 4-O-caffeoylquinic acid (4-CQA), a chlorogenic acid derivative, on changes in the expression of SOX9 (SRY-box transcription factor 9), ACAN (aggrecan), and COL2A1 (collagen type II alpha 1 chain) genes in chondrocytes.

[0020] Figure 4b shows the results of analyzing the effect of 4-O-caffeoylquinic acid (4-CQA), a chlorogenic acid derivative, on changes in the expression of SOX9 (SRY-box transcription factor 9) and ACAN (aggrecan) proteins in chondrocytes.

[0021] Figure 5 shows the results of evaluating the cytotoxicity of 5-O-caffeoylquinic acid (5-CQA), a chlorogenic acid derivative, in chondrocytes.

[0022] Figure 6a shows the results of analyzing the effect of 5-O-caffeoylquinic acid (5-CQA), a chlorogenic acid derivative, on the expression of PTGS2 (prostaglandin-endoperoxide synthase 2) and NOS2 (nitric oxide synthase 2) genes, which are inflammation-related factors, in chondrocytes.

[0023] Figure 6b shows the results of analyzing the effect of 5-O-caffeoylquinic acid (5-CQA), a chlorogenic acid derivative, on changes in the expression of inflammatory factors COX2 (cyclooxygenase 2) and NOS2 (nitric oxide synthase 2) proteins in chondrocytes.

[0024] Figure 7a shows the results of analyzing the effect of 5-O-caffeoylquinic acid (5-CQA), a chlorogenic acid derivative, on the expression of MMP3 (matrix metalloproteinase-3), MMP13 (matrix metalloproteinase-13), and ADAMTS5 (ADAM metallopeptidase with thrombospondin type 1 motif 5) genes in chondrocytes.

[0025] Figure 7b shows the results of analyzing the effect of 5-O-caffeoylquinic acid (5-CQA), a chlorogenic acid derivative, on changes in the expression of matrix metalloproteinase-3 (MMP3) and matrix metalloproteinase-13 (MMP13) proteins in chondrocytes.

[0026] Figure 7c shows the results of analyzing the effect of 5-O-caffeoylquinic acid (5-CQA), a chlorogenic acid derivative, on the levels of matrix metalloproteinase-3 (MMP3) and matrix metalloproteinase-13 (MMP13) proteins secreted outside the cells in chondrocytes.

[0027] Figure 8a shows the results of analyzing the effect of 5-O-caffeoylquinic acid (5-CQA), a chlorogenic acid derivative, on changes in the expression of SOX9 (SRY-box transcription factor 9), ACAN (aggrecan), and COL2A1 (collagen type II alpha 1 chain) genes in chondrocytes.

[0028] Figure 8b shows the results of analyzing the effect of 5-O-caffeoylquinic acid (5-CQA), a chlorogenic acid derivative, on changes in the expression of SOX9 (SRY-box transcription factor 9) and ACAN (aggrecan) proteins in chondrocytes.

[0029] Figure 9 shows the results of evaluating the cytotoxicity of 1,3-O-caffeoylquinic acid (1,3-DiCQA), a chlorogenic acid derivative, in chondrocytes.

[0030] Figure 10a shows the results of analyzing the effect of 1,3-O-caffeoylquinic acid (1,3-DiCQA), a chlorogenic acid derivative, on the expression of PTGS2 (prostaglandin-endoperoxide synthase 2) and NOS2 (nitric oxide synthase 2) genes, which are inflammation-related factors, in chondrocytes.

[0031] Figure 10b shows the results of a quantitative analysis of the effect of 1,3-O-caffeoylquinic acid (1,3-DiCQA), a chlorogenic acid derivative, on the expression of PTGS2 (prostaglandin-endoperoxide synthase 2) and NOS2 (nitric oxide synthase 2) genes, which are inflammation-related factors, in chondrocytes.

[0032] Figure 10c shows the results of analyzing the effect of 1,3-O-caffeoylquinic acid (1,3-DiCQA), a chlorogenic acid derivative, on changes in the expression of inflammatory factors COX2 (cyclooxygenase 2) and NOS2 (nitric oxide synthase 2) proteins in chondrocytes.

[0033] Figure 11a shows the results of analyzing the effect of 1,3-O-caffeoylquinic acid (1,3-DiCQA), a chlorogenic acid derivative, on changes in the expression of MMP3 (matrix metalloproteinase-3), MMP13 (matrix metalloproteinase-13), and ADAMTS5 (ADAM metallopeptidase with thrombospondin type 1 motif 5) genes in chondrocytes.

[0034] Figure 11b shows the results of a quantitative analysis of the effect of 1,3-O-caffeoylquinic acid (1,3-DiCQA), a chlorogenic acid derivative, on the expression of MMP3 (matrix metalloproteinase-3), MMP13 (matrix metalloproteinase-13), and ADAMTS5 (ADAM metallopeptidase with thrombospondin type 1 motif 5) genes in chondrocytes.

[0035] Figure 11c shows the results of analyzing the effect of 1,3-O-caffeoylquinic acid (1,3-DiCQA), a chlorogenic acid derivative, on changes in the expression of matrix metalloproteinase-3 (MMP3) and matrix metalloproteinase-13 (MMP13) proteins in chondrocytes.

[0036] Figure 11d shows the results of analyzing the effect of 1,3-O-caffeoylquinic acid (1,3-DiCQA), a chlorogenic acid derivative, on the levels of MMP3 (matrix metalloproteinase-3) and MMP13 (matrix metalloproteinase-13) proteins secreted outside the cells in chondrocytes.

[0037] Figure 12a shows the results of analyzing the effect of 1,3-O-caffeoylquinic acid (1,3-DiCQA), a chlorogenic acid derivative, on changes in the expression of SOX9 (SRY-box transcription factor 9), ACAN (aggrecan), and COL2A1 (collagen type II alpha 1 chain) genes in chondrocytes.

[0038] Figure 12b shows the results of a quantitative analysis of the effect of 1,3-O-caffeoylquinic acid (1,3-DiCQA), a chlorogenic acid derivative, on changes in the expression of SOX9 (SRY-box transcription factor 9), ACAN (aggrecan), and COL2A1 (collagen type II alpha 1 chain) genes in chondrocytes.

[0039] Figure 12c shows the results of analyzing the effect of 1,3-O-caffeoylquinic acid (1,3-DiCQA), a chlorogenic acid derivative, on changes in the expression of SOX9 (SRY-box transcription factor 9) and ACAN (aggrecan) proteins in chondrocytes.

[0040] Figure 13 is a schematic diagram showing the mechanism of action of chlorogenic acid derivatives in treating osteoarthritis.

[0041] The terms used in this specification have been selected from widely used, current terms, taking into account the functions of the present invention. However, these terms may vary depending on the intentions of those skilled in the art, precedents, the emergence of new technologies, etc. Furthermore, in certain cases, terms may be arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this invention should not be defined simply as names, but rather based on their inherent meanings and the overall content of the present invention.

[0042] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0043] Hereinafter, the present invention will be described in more detail.

[0044] The present invention provides a pharmaceutical composition for preventing or treating osteoarthritis, comprising a chlorogenic acid derivative, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof as an active ingredient.

[0045] The above chlorogenic acid is also named 3-O-caffeoylquinic acid and is a compound represented by the following chemical formula 1, and its IUPAC name is (1S,3R,4R,5R)-3-[(E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy-1,4,5-trihydroxycyclohexane-1-carboxylic acid.

[0046] [Chemical Formula 1]

[0047]

[0048] The above chlorogenic acid derivative is 4-O-caffeoylquinic acid (4-CQA). 4-O-caffeoylquinic acid (4-CQA), a chlorogenic acid derivative, is also called cryptochlorogenic acid and is a compound represented by the following chemical formula 2, and its IUPAC name is (3R,5R)-4-[(E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy-1,3,5-trihydroxycyclohexane-1-carboxylic acid.

[0049] [Chemical Formula 2]

[0050]

[0051] The above chlorogenic acid derivative is 5-O-caffeoylquinic acid (5-CQA). 5-O-Caffeoylquinic acid (5-CQA), a chlorogenic acid derivative, is also called neochlorogenic acid and is a compound represented by the following chemical formula 3. Its IUPAC name is (1R,3R,4S,5R)-3-[(E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy-1,4,5-trihydroxycyclohexane-1-carboxylic acid.

[0052] [Chemical Formula 3]

[0053]

[0054] The above chlorogenic acid derivative is 1,3-O-caffeoylquinic acid (1,3-DiCQA). 1,3-O-Caffeoylquinic acid (1,3-DiCQA), a chlorogenic acid derivative, is a compound represented by the following chemical formula 4, and its IUPAC name is (1R,3R,4S,5R)-3-[(E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy-1,4,5-trihydroxycyclohexane-1-carboxylic acid ((1S,3R,4R,5R)-1,3-bis[[(E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy]-4,5-dihydroxycyclohexane-1-carboxylic acid).

[0055] [Chemical Formula 4]

[0056]

[0057] The above chlorogenic acid derivative inhibits the expression of PTGS2 (prostaglandin-endoperoxide synthase 2), NOS2 (nitric oxide synthase 2), MMP3 (matrix metalloproteinase-3), MMP13 (matrix metalloproteinase-13), and ADAMTS5 (ADAM metallopeptidase with thrombospondin type 1 motif 5) genes.

[0058] In addition, the chlorogenic acid derivative inhibits the expression of COX2 (cyclooxygenase 2), NOS2 (nitric oxide synthase 2), MMP3 (matrix metalloproteinase-3), and MMP13 (matrix metalloproteinase-13) proteins.

[0059] The above hydrate means a compound and water bound by non-covalent intermolecular forces, and includes a stoichiometric or non-stoichiometric amount of water. Specifically, the hydrate may include water in a ratio of about 0.25 mol to about 10 mol based on 1 mol of the active ingredient, and more specifically, may include about 0.5 mol, about 1 mol, about 1.5 mol, about 2 mol, about 3 mol, about 5 mol, etc.

[0060] The above solvate means a compound and a solvent bound by non-covalent intermolecular forces, and includes a stoichiometric or non-stoichiometric amount of the solvent. Preferred solvents are volatile, non-toxic, and can be administered to humans in very small amounts. Specifically, the solvate may contain water in a ratio of about 0.25 mol to about 10 mol based on 1 mol of the active ingredient, and more specifically, may contain about 0.5 mol, about 1 mol, about 1.5 mol, about 2 mol, about 3 mol, about 5 mol, etc.

[0061] The above pharmaceutically acceptable salt means an acid addition salt formed by a pharmaceutically acceptable free acid, and the pharmaceutically acceptable salt means a salt commonly used in the pharmaceutical industry, for example, an inorganic ion salt manufactured with calcium, potassium, sodium or magnesium, etc.; an inorganic acid salt manufactured with hydrochloric acid, nitric acid, phosphoric acid, hydrobromic acid, iodic acid, perchloric acid or sulfuric acid, etc.; an organic acid salt manufactured with acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid or vanillic acid, etc.; There are sulfonic acid salts manufactured from methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, etc.; amino acid salts manufactured from glycine, arginine, lysine, etc.; or amine salts manufactured from trimethylamine, triethylamine, ammonia, pyridine, picoline, etc.; but the types of salts meant in the present invention are not limited by these listed salts.

[0062] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains.

[0063] The pharmaceutically acceptable carriers mentioned above are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

[0064] In the present invention, the content of the additive included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.

[0065] The above pharmaceutical composition may be formulated in the form of one or more external preparations selected from the group consisting of injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, and cataplasmas.

[0066] The pharmaceutical composition of the present invention may further comprise pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include, but are not limited to, excipients such as starches, sugars, and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose; binders such as gelatin, alginates, and polyvinyl pyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol; disintegrants such as povidone and crospovidone; and surfactants such as polysorbates, cetyl alcohol, and glycerol. The pharmaceutically acceptable carriers and diluents may be biologically and physiologically compatible with the subject. Examples of diluents include, but are not limited to, saline, aqueous buffers, solvents, and / or dispersion media.

[0067] The pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. In the case of oral administration, it may be formulated as tablets, troches, lozenges, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, or elixirs. In the case of parenteral administration, it may be formulated as injections, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for application, oils, creams, etc.

[0068] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition and weight, age, sex, health status, dietary constitution, nature of the formulation, severity of the disease, administration time of the composition, administration method, administration period or interval, excretion rate, and drug form, and may be appropriately selected by a person skilled in the art. For example, the dosage may range from about 0.1 to 10,000 mg / kg, but is not limited thereto, and may be administered once or several times a day in divided doses.

[0069] The pharmaceutical composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. The pharmaceutically effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration method, administration time, and / or administration route of the pharmaceutical composition, and a person skilled in the art can easily determine and prescribe an effective dosage for the intended treatment. The pharmaceutical composition of the present invention may be administered once a day or divided into several doses.

[0070] In addition, the present invention provides a health functional food composition for preventing or improving osteoarthritis, comprising a chlorogenic acid derivative, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof as an active ingredient.

[0071] The present invention can be generally used as a commonly used food.

[0072] The food composition of the present invention can be used as a health functional food. The term "health functional food" refers to a food manufactured and processed using raw materials or ingredients with functional properties beneficial to the human body, as defined by the Health Functional Food Act. "Functionality" refers to ingestion for the purpose of obtaining beneficial effects for health purposes, such as regulating nutrients for the structure and functions of the human body or physiological effects.

[0073] The food composition of the present invention may include conventional food additives, and its suitability as the "food additive" is determined by the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additive Code approved by the Ministry of Food and Drug Safety, unless otherwise specified.

[0074] Items listed in the above "Food Additives Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high-molecular-weight pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents for noodles, preservative preparations, and tar color preparations.

[0075] The food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc.

[0076] For example, among health functional foods in capsule form, hard capsules can be manufactured by mixing and filling a composition according to the present invention with additives such as excipients into a conventional hard capsule, and soft capsules can be manufactured by mixing the composition according to the present invention with additives such as excipients and filling it into a capsule base such as gelatin. The soft capsules may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc., as needed.

[0077] The definitions of terms for the above excipients, binders, disintegrants, lubricants, flavoring agents, etc. are described in literature known in the art and include those with identical or similar functions. There are no specific restrictions on the type of food, and all health functional foods in the conventional sense are included.

[0078] As used herein, the term "prevention" refers to any action that suppresses or delays the onset of a disease by administering a composition according to the present invention. The term "treatment" refers to any action that improves or beneficially alters the symptoms of a disease by administering a composition according to the present invention. As used herein, "improvement" refers to any action that improves the worsening condition of a disease by administering or ingesting a composition according to the present invention to a subject.

[0079] Hereinafter, to aid understanding of the present invention, experimental examples and examples will be described in detail. However, the following experimental examples and examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental examples and examples of the present invention are provided to more fully explain the present invention to those of average skill in the art.

[0080]

[0081] <Experimental Example> Experimental Materials and Methods

[0082] The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

[0083]

[0084] 1. Isolation and culture of articular chondrocytes from mouse cartilage

[0085] Articular cartilage tissue was harvested from the knee area of ​​4-day-old mice, and then digested with 0.2% collagenase and 0.25% trypsin. Articular chondrocytes were obtained from the digested articular cartilage tissue. The obtained articular chondrocytes were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS).

[0086]

[0087] 2. Expression of genes related to inflammation and cartilage degeneration in IL-1β-induced osteoarthritic chondrocytes

[0088] The obtained mouse articular chondrocytes were cultured in a 35 cm 2 3.0 X 10 in a culture dish 5 Cells were seeded at a concentration of 100 cells and cultured for 2 days. After 2 days of culture, the medium was replaced, and the cells were treated with chlorogenic acid derivatives 4-CQA (caffeoylquinic acid), 5-CQA, and 1,3-DiCQA for 1 hour, and IL-1β was treated at a concentration of 1 ng / mL to induce osteoarthritis. After culturing for 36 hours, RNA was extracted using RNAiso plus (Takara) reagent, and cDNA was synthesized using reverse transcriptase. PCR and qRT-PCR were performed using the synthesized cDNA.

[0089]

[0090] 3. Protein expression analysis using Western blot

[0091] After culturing the obtained mouse articular chondrocytes, total cell lysates were obtained using RIPA (Radio-Immunoprecipitation Assay) buffer. Proteins were quantified using the BCA Protein Assay Kit. Equal amounts of protein were electrophoresed using SDS-PAGE and transferred to nitrocellulose membranes. Protein expression was compared and analyzed using the respective primary antibodies, and a secondary antibody conjugated with HRP (Horseradish peroxidase) was used.

[0092] Additionally, the release of matrix metalloproteinase-3 (MMP3) and matrix metalloproteinase-13 (MMP13) proteins was measured. After culturing mouse articular chondrocytes as described above, the proteins contained in the culture medium were precipitated with trichloroacetic acid. The precipitated proteins were analyzed by Western blotting to confirm the MMP3 and MMP13 proteins released outside the cells.

[0093]

[0094] Example 1. Inhibition of the expression of genes and proteins related to inflammation and joint degeneration by 4-CQA.

[0095] According to the present invention, the therapeutic effect of 4-O-caffeoylquinic acid (4-CQA), a chlorogenic acid derivative, on IL-1β-induced osteoarthritis in chondrocytes was evaluated by changes in the expression levels of genes and proteins related to joint degeneration.

[0096] First, the cytotoxicity of 4-CQA was evaluated. Chondrocytes were treated with 4-CQA at concentrations of 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, and cell viability was analyzed after 24 or 36 hours. As shown in Figure 1, chondrocytes were confirmed to exhibit no cytotoxicity even when treated with 4-CQA at concentrations up to 100 μM.

[0097] The expression levels of PTGS2 (prostaglandin-endoperoxide synthase 2) and NOS2 (nitric oxide synthase 2), which are inflammation-related factors, were analyzed by RT-PCR and qRT-PCR, and the expression levels of COX2 (cyclooxygenase 2) and iNOS2 proteins were analyzed. The PTGS2 gene is the mRNA of COX2 (cyclooxygenase 2), which produces PGE2 (prostaglandin 2) in the inflammatory response, and iNOS plays a role in producing nitric oxide (NO) in the inflammatory response.

[0098] The effect of 4-CQA on the expression of PTGS2 and NOS2 genes in mouse articular chondrocytes was analyzed. As shown in Figure 2a, when mouse articular chondrocytes were treated with IL-1β, the expression of PTGS2 and NOS2 genes increased. As a result of treatment with 4-CQA, the expression of PTGS2 and NOS2 genes increased by IL-1β was confirmed to be suppressed. In addition, the effect of 4-CQA on the expression of COX2 and NOS2 proteins in mouse articular chondrocytes was analyzed. As shown in Figure 2b, when mouse articular chondrocytes were treated with IL-1β, the expression of COX2 and NOS2 proteins increased. As a result of treatment with 4-CQA, the expression of COX2 and NOS2 proteins increased by IL-1β was confirmed to be suppressed.

[0099] We analyzed the release of matrix metalloproteinase-3 (MMP3) and matrix metalloproteinase-13 (MMP13) proteins from chondrocytes. MMP3 and MMP13 proteins are types of matrix metalloproteinases, and their expression increases when osteoarthritis is induced, degrading the extracellular matrix and thus degrading articular cartilage.

[0100] The effect of 4-CQA on changes in MMP3 and MMP13 gene expression in mouse articular chondrocytes was analyzed. Furthermore, the effect of 4-CQA on changes in MMP3 and MMP13 protein expression in mouse articular chondrocytes was analyzed. As shown in Figures 3A and 3B, treatment of mouse articular chondrocytes with IL-1β increased the gene and protein expression of MMP3 and MMP13. Treatment with 4-CQA suppressed the IL-1β-induced increase in MMP3 and MMP13 gene and protein expression. Furthermore, the effect of 4-CQA on the levels of MMP3 and MMP13 proteins secreted into the extracellular space in mouse articular chondrocytes was analyzed. As shown in Fig. 3c, the levels of MMP3 and MMP13 in the extracellular matrix decomposition proteins secreted outside of chondrocytes were confirmed to increase when IL-1β was treated, but to decrease when 4-CQA was treated.

[0101] COL2A1 (collagen type II alpha 1 chain) and ACAN (aggrecan) are representative extracellular matrix proteins that constitute stromal cartilage and play a crucial role in maintaining cartilage homeostasis. SOX9 (SRY-box transcription factor 9) is a factor that regulates the expression of COL2A1 and ACAN proteins. As osteoarthritis progresses, SOX9 expression decreases, and along with this, extracellular matrix proteins decrease due to the action of enzymes that degrade the extracellular matrix.

[0102] The effect of 4-CQA on the expression of SOX9, ACAN, and COL2A1 genes in mouse articular chondrocytes was analyzed. As shown in Figure 4a, when mouse articular chondrocytes were treated with IL-1β, the gene expression of SOX9, ACAN, and COL2A1 decreased. As a result of treatment with 4-CQA, the gene expression of SOX9, ACAN, and COL2A1 decreased due to IL-1β was restored. In addition, the effect of 4-CQA on the protein expression of SOX9 and ACAN in mouse articular chondrocytes was analyzed. As shown in Figure 4b, when mouse articular chondrocytes were treated with IL-1β, the protein expression of SOX9 and ACAN, which are extracellular matrix proteins degraded by MMPs and ADAMTs, was decreased. As a result of treatment with 4-CQA, the protein expression of SOX9 and ACAN decreased due to IL-1β was restored.

[0103] The above results demonstrate that 4-O-caffeoylquinic acid (4-CQA), a chlorogenic acid derivative, suppresses the expression of inflammatory factors in IL-1β-induced osteoarthritis-related chondrocytes, suppresses the expression of extracellular matrix degrading factors, and restores the expression of cartilage homeostasis-related factors, thereby exhibiting preventive and therapeutic effects on osteoarthritis.

[0104]

[0105] Example 2. Inhibition of the expression of genes and proteins related to inflammation and joint degeneration by 5-CQA.

[0106] According to the present invention, the therapeutic effect of 5-O-caffeoylquinic acid (5-CQA), a chlorogenic acid derivative, on IL-1β-induced osteoarthritis in chondrocytes was evaluated by changes in the expression levels of genes and proteins related to joint degeneration.

[0107] First, the cytotoxicity of 5-CQA was evaluated. Chondrocytes were treated with 5-CQA at concentrations of 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, and cell viability was analyzed after 24 or 36 hours. As shown in Figure 5, chondrocytes were confirmed to exhibit no cytotoxicity even when treated with 5-CQA at concentrations up to 100 μM.

[0108] The effect of 5-CQA on the expression of PTGS2 and NOS2 genes in mouse articular chondrocytes was analyzed. As shown in Figure 6a, when mouse articular chondrocytes were treated with IL-1β, the expression of PTGS2 and NOS2 genes increased. As a result of treatment with 5-CQA, the expression of PTGS2 and NOS2 genes increased by IL-1β was confirmed to be suppressed. In addition, the effect of 5-CQA on the expression of COX2 and NOS2 proteins in mouse articular chondrocytes was analyzed. As shown in Figure 6b, when mouse articular chondrocytes were treated with IL-1β, the expression of COX2 and NOS2 proteins increased. As a result of treatment with 5-CQA, the expression of COX2 and NOS2 proteins increased by IL-1β was confirmed to be suppressed.

[0109] The effects of 5-CQA on the expression of MMP3, MMP13, and ADAMTS5 (ADAM metallopeptidase with thrombospondin type 1 motif 5) genes in mouse articular chondrocytes were analyzed. As shown in Figure 7a, when mouse articular chondrocytes were treated with IL-1β, the expression of MMP3, MMP13, and ADAMTS5 genes increased. As a result of treatment with 5-CQA, it was confirmed that the IL-1β-induced expression of MMP3, MMP13, and ADAMTS5 genes was suppressed. In addition, the effects of 5-CQA on the expression of MMP3 and MMP13 proteins in mouse articular chondrocytes were analyzed. As shown in Figure 7b, when mouse articular chondrocytes were treated with IL-1β, the expression of MMP3 and MMP13 proteins increased. As a result of treatment with 5-CQA, it was confirmed that the expression of MMP3 and MMP13 proteins increased by IL-1β was suppressed. The effect of 5-CQA on the levels of MMP3 and MMP13 proteins secreted into the extracellular matrix in mouse articular chondrocytes was analyzed. As shown in Figure 7c, the levels of MMP3 and MMP13 in the extracellular matrix degrading proteins secreted into the extracellular matrix of chondrocytes increased when treated with IL-1β, but decreased when treated with 5-CQA.

[0110] The effect of 5-CQA on the expression of COL2A1, ACAN, and SOX9 genes in mouse articular chondrocytes was analyzed. As shown in Figure 8a, when IL-1β was treated in mouse articular chondrocytes, the gene expression of SOX9, ACAN, and COL2A1 was decreased. As a result of 5-CQA treatment, it was confirmed that the gene expression of SOX9, ACAN, and COL2A1 decreased by IL-1β was restored. In addition, the effect of 5-CQA on the protein expression of SOX9 and ACAN in mouse articular chondrocytes was analyzed. As shown in Figure 8b, when IL-1β was treated in mouse articular chondrocytes, the protein expression of SOX9 and ACAN was decreased. As a result of 5-CQA treatment, it was confirmed that the protein expression of SOX9 and ACAN decreased by IL-1β was restored.

[0111] The above results demonstrate that 5-O-caffeoylquinic acid (5-CQA), a chlorogenic acid derivative, exhibits preventive and therapeutic effects on osteoarthritis by inhibiting the expression of inflammatory factors in IL-1β-induced osteoarthritis-related chondrocytes, inhibiting the expression of extracellular matrix degrading factors, and restoring the expression of cartilage homeostasis-related factors.

[0112]

[0113] Example 3. Inhibition of the expression of genes and proteins related to inflammation and joint degeneration by 1,3-DiCQA.

[0114] According to the present invention, the therapeutic effect of 1,3-O-caffeoylquinic acid (1,3-DiCQA), a chlorogenic acid derivative, on IL-1β-induced osteoarthritis in chondrocytes was evaluated by changes in the expression levels of genes and proteins related to joint degeneration.

[0115] First, the cytotoxicity of 1,3-DiCQA was evaluated. Chondrocytes were treated with 1,3-DiCQA at concentrations of 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, and cell viability was analyzed after 24 or 48 hours. As shown in Figure 9, it was confirmed that chondrocytes did not exhibit cytotoxicity even when treated with 1,3-DiCQA at concentrations up to 100 μM.

[0116] Chondrocytes were treated with 1,3-DiCQA at concentrations of 12.5 μM, 25 μM, 50 μM, and 100 μM, and then treated with IL-1β 1 hour later and cultured for 36 hours to induce osteoarthritis. The effect of 1,3-DiCQA on the expression of PTGS2 and NOS2 genes in mouse articular chondrocytes was analyzed. As shown in Fig. 10a, when mouse articular chondrocytes were treated with IL-1β, the expression of PTGS2 and NOS2 genes increased. As a result of treatment with 1,3-DiCQA, it was confirmed that the expression of PTGS2 and NOS2 genes increased by IL-1β was suppressed. In addition, as a result of quantitative analysis of the changes in the expression of the PTGS2 and NOS2 genes, as shown in Fig. 10b, it was confirmed that the expression of the PTGS2 and NOS2 genes decreased in a concentration-dependent manner as the concentration of 1,3-DiCQA treatment increased. In particular, when 1,3-DiCQA was treated at a concentration of 100 μM, the expression levels of the PTGS2 and NOS2 genes were confirmed to decrease to a level similar to that of the untreated control group (None). In addition, the effect of 1,3-DiCQA on the changes in the expression of COX2 and NOS2 proteins in mouse articular chondrocytes was analyzed. As shown in Fig. 10c, when IL-1β was treated in mouse articular chondrocytes, the expression of COX2 and NOS2 proteins increased. As a result of treatment with 1,3-DiCQA, it was confirmed that the expression of COX2 and NOS2 proteins increased by IL-1β was suppressed.

[0117] The effect of 1,3-DiCQA on the expression of MMP3, MMP13, and ADAMTS5 genes in mouse articular chondrocytes was analyzed. As shown in Figures 11a and 11b, when mouse articular chondrocytes were treated with IL-1β, the expression of MMP3, MMP13, and ADAMTS5 genes increased. As a result of treatment with 1,3-DiCQA, it was confirmed that the expression of MMP3, MMP13, and ADAMTS5 genes increased by IL-1β was suppressed in a 1,3-DiCQA treatment dose-dependent manner. In addition, the effect of 1,3-DiCQA on the expression of MMP3 and MMP13 proteins in mouse articular chondrocytes was analyzed. As shown in Figure 11c, when mouse articular chondrocytes were treated with IL-1β, the expression of MMP3 and MMP13 proteins increased. Treatment with 1,3-DiCQA confirmed that the expression of MMP3 and MMP13 proteins increased by IL-1β was suppressed. The effect of 5-CQA on the levels of MMP3 and MMP13 proteins secreted into the extracellular matrix of mouse articular chondrocytes was analyzed. As shown in Figure 11d, the levels of MMP3 and MMP13 in the extracellular matrix degrading proteins secreted into the extracellular matrix of chondrocytes increased when treated with IL-1β, but decreased when treated with 1,3-DiCQA.

[0118] The effect of 1,3-DiCQA on the expression of COL2A1, ACAN, and SOX9 genes in mouse articular chondrocytes was analyzed. As shown in Figures 12a and 12b, when mouse articular chondrocytes were treated with IL-1β, the expression of the genes SOX9, ACAN, and COL2A1 decreased. As a result of treatment with 1,3-DiCQA, it was confirmed that the gene expression of SOX9, ACAN, and COL2A1 decreased by IL-1β was restored in a 1,3-DiCQA treatment dose-dependent manner. In addition, the effect of 1,3-DiCQA on the protein expression of SOX9 and ACAN in mouse articular chondrocytes was analyzed. As shown in Figure 12c, when mouse articular chondrocytes were treated with IL-1β, the protein expression of SOX9 and ACAN decreased. As a result of treatment with 1,3-DiCQA, it was confirmed that the protein expression of SOX9 and ACAN, which had been reduced by IL-1β, was restored.

[0119] The above results demonstrate that 1,3-O-caffeoylquinic acid (1,3-DiCQA), a chlorogenic acid derivative, suppresses the expression of inflammatory factors in IL-1β-induced osteoarthritis-related chondrocytes, suppresses the expression of extracellular matrix degrading factors, and restores the expression of cartilage homeostasis-related factors, thereby exhibiting preventive and therapeutic effects on osteoarthritis.

[0120] In conclusion, among chlorogenic acid derivatives, 4-O-caffeoylquinic acid (4-CQA), 5-O-caffeoylquinic acid (5-CQA), and 1,3-O-caffeoylquinic acid (1,3-DiCQA) were found to have preventive and therapeutic effects on osteoarthritis by suppressing the expression of inflammatory factors, suppressing the expression of extracellular matrix degrading factors, and restoring the expression of cartilage homeostasis-related factors in osteoarthritis-induced chondrocytes (Fig. 13).

[0121] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0122] Numerical ranges are inclusive of the values ​​defined in the ranges above. Any maximum numerical limitation given throughout this specification includes any lower numerical limitation, as if that lower numerical limitation were explicitly stated. Any minimum numerical limitation given throughout this specification includes any higher numerical limitation, as if that higher numerical limitation were explicitly stated. Any numerical limitation given throughout this specification will include any better numerical range within the broader numerical range, as if that narrower numerical limitation were explicitly stated.

Claims

1. A pharmaceutical composition for preventing or treating osteoarthritis, comprising a chlorogenic acid derivative, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof as an active ingredient.

2. A pharmaceutical composition for preventing or treating osteoarthritis, characterized in that the chlorogenic acid derivative in paragraph 1 is 4-O-caffeoylquinic acid (4-CQA) represented by the following chemical formula 2. [Chemical Formula 2] 3. A pharmaceutical composition for preventing or treating osteoarthritis, characterized in that the chlorogenic acid derivative in paragraph 1 is 5-O-caffeoylquinic acid (5-CQA) represented by the following chemical formula 3. [Chemical Formula 3] 4. A pharmaceutical composition for preventing or treating osteoarthritis, characterized in that the chlorogenic acid derivative in paragraph 1 is 1,3-O-caffeoylquinic acid (1,3-DiCQA) represented by the following chemical formula 4. [Chemical Formula 4] 5. A pharmaceutical composition for preventing or treating osteoarthritis, characterized in that the chlorogenic acid derivative of paragraph 1 inhibits the expression of PTGS2 (prostaglandin-endoperoxide synthase 2), NOS2 (nitric oxide synthase 2), MMP3 (matrix metalloproteinase-3), MMP13 (matrix metalloproteinase-13), and ADAMTS5 (ADAM metallopeptidase with thrombospondin type 1 motif 5) genes.

6. A pharmaceutical composition for preventing or treating osteoarthritis, characterized in that the chlorogenic acid derivative in paragraph 1 inhibits the expression of COX2 (cyclooxygenase 2), iNOS2 (inducible nitric oxide synthase 2), MMP3 (matrix metalloproteinase-3), and MMP13 (matrix metalloproteinase-13) proteins.

7. A health functional food composition for preventing or improving osteoarthritis, comprising a chlorogenic acid derivative, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof as an active ingredient.

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